Method for detecting post-translation modifications of peptides

ABSTRACT

Method is described for sequencing polypeptides by forming peptide ladders comprising a series of polypeptides in which adjacent members of the series vary by one amino acid residue and determining the identity and position of each amino acid in the polypeptide by mass spectroscopy.

RELATED APPLICATION

[0001] This application is a continuation in part of copending andcommonly owned application Ser. No. 07/891,177 filed May 29, 1992.

FIELD OF THE INVENTION

[0002] This invention relates to rapid and efficient methods forsequencing formed or forming polypeptides utilizing a mass spectrometer.

[0003] Polypeptides are a class of compounds composed of α-amino acidresidues chemically bonded together by amide linkages with eliminationof water between the carboxy group of one amino acid and the amino groupof another amino acid. A polypeptide is thus a polymer of α-amino acidresidues which may contain a large number of such residues. Peptides aresimilar to polypeptides, except that they are comprised of a lessernumber of α-amino acids. There is no clear-cut distinction betweenpolypeptides and peptides. For convenience, in this disclosure andclaims, the term “polypeptide” will be used to refer generally topeptides and polypeptides.

[0004] Proteins are polypeptide chains folded into a defined threedimensional structure. They are complex high polymers containing carbon,hydrogen, nitrogen, and sulfur and are comprised of linear chains ofamino acids connected by peptide links. They are similar topolypeptides, but of a much higher molecular weight.

[0005] For a complete understanding of physiological reactions involvingproteins it is often necessary to understand their structure. There area number of facets to the structure of proteins. These are the primarystructure which is concerned with amino acid sequence in the proteinchain and the secondary, tertiary and quaternary structures whichgenerally relate to the three dimensional configuration of proteins.This invention is concerned with sequencing polypeptides to assist indetermining the primary structure of proteins. It provides a facile andaccurate procedure for sequencing polypeptides. It is also applicable tosequencing the amino acid residues at the termini of proteins.

[0006] Many procedures have been used over the years to determine theamino acid sequence, i.e. the primary structure, of polypeptides andproteins. At the present time, the best method available for suchdeterminations is the Edman degradation. In this procedure, one aminoterminal amino acid residue at a time is removed from a polypeptide tobe analyzed. That amino acid is normally identified by reverse phasehigh performance liquid chromatography (HPLC), but recently massspectrometric procedures have been described for this purpose (1) TheEdman degradation cycle is repeated for each successive terminal aminoacid residue until the complete polypeptide has been degraded. Theprocedure is tedious and time consuming. Each sequential removal of aterminal amino acid requires 20 to 30 minutes. Hence, with a polypeptideof even moderate length, say for example 50 amino acid residues, asequence determination may require many hours. The procedure has beenautomated. The automated machines are available as sequenators, but itstill requires an unacceptable amount of time to carry out a sequenceanalysis. Although the procedure is widely employed, one which requiredless time and which yielded information about a broader range ofmodified or unusual amino acid residues present in a polypeptide wouldbe very useful to the art. A process which can be used to sequenceindividual members of mixtures of polypeptides would be particularlyuseful.

[0007] Recent advances in the art of mass spectroscopy have made itpossible to obtain characterizing data from extremely small amounts ofpolypeptide samples. It is, for example, presently possible because ofthe sensitivity and precision of available instruments to obtain usefuldata utilizing from picomole to subpicomole amounts of products to beanalyzed. Further, the incipient ion-trap technologies promise evenbetter sensitivities, and have already been demonstrated to yield usefulspectra in the 10⁻15 to 10⁻16 sample range.

[0008] In general, both electrospray and matrix-assisted laserdesorption ionizaton methods mainly generate intact molecular ions. Theresolution of the electrospray quadrupole instruments is about 1 in2,000 and that of the laser desorption time-of-flight instruments about1 in 400. Both techniques give mass accuracies of about 1 in 10-20,000(i.e. +/−0.01% or better). There are proposed modifications oftime-of-flight analyzer that may improve the resolution by up to factorof 10-fold, and markedly improve the sensitivity of that technique.

[0009] These techniques yield mass measurements accurate to +/−0.2atomic mass units, or better. These capabilities mean that, by employingthe process of this invention, the polypeptide itself whether alreadyformed or as it is being formed can be sequenced more readily, withgreater speed, sensitivity, and precision, than the amino acidderivative released by stepwise degradation techniques such as the Edmandegradation. As will be explained in more detail below, the process ofthis invention employs a novel technique of sequence determination inwhich a mixture containing a family of fragments, each differing by asingle amino acid residue is produced and thereafter analyzed by massspectroscopy.

SUMMARY OF THE INVENTION

[0010] This invention provides a method for the sequential analysis ofpolypeptides which may be already formed or are being formed byproducing under controlled conditions, from the formed polypeptide orfrom the segments of the polypeptide as it is being formed, a mixturecontaining a series of adjacent polypeptides in which each member of theseries differs from the next adjacent member by one amino acid residue.The mixture is then subjected to mass spectrometric analysis to generatea spectrum in which the peaks represent the separate members of theseries. The differences in molecular mass between such adjacent memberscoupled with the position of the peaks in the spectrum for such adjacentmembers is indicative of the identity of the said amino acid residue andof its position in the chain of the formed or forming polypeptide.

[0011] The process of this invention which utilizes controlled cyclingof reaction conditions to produce peptide ladders of predictablestructure is to be contrasted with previous methods employing massspectroscopy including exopeptidase digestion on uncontrolled chemicaldegradation. See references 2-5. Because of the uncontrolled nature ofthese previous methods, only incomplete sequence information could beobtained.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 indicates a family or mixture of polypeptides (peptideladder, as defined hereinafter) derived from a single formed polypeptidecontaining n amino acid residues. The mixture is analyzed in accordancewith this invention to determine the amino acid sequence of the originalpolypeptide. Each amino acid in the sequence is denoted by a number withthe numbering starting at the amino terminal of the peptide. X denotes aterminating group.

[0013]FIG. 2 is an idealized mass spectrum of the peptide ladder of apolypeptide similar to the family shown in FIG. 1.

[0014]FIG. 3 shows the reactions involved in generating a peptide ladderfrom a formed polypeptide for analysis utilizing phenyl isothiocyanate(PITC) as the coupling reagent and phenyl isocyanate (PIC) as theterminating reagent.

[0015]FIG. 4 is a more precise summary of the process shown in FIG. 3.

[0016]FIG. 5 is an idealized mass spectrum of peptide ladders obtainedfrom a mixture of two formed polypeptides one of which is identified asA, the other as B.

[0017]FIG. 6 is a positive ion, matrix assisted laser desorption massspectrum of the formed polypeptide [Glu¹]fibrinopeptide B.

[0018]FIG. 7 is a positive ion matrix assisted laser desorption spectrumof [Glu¹]fibrinopeptide B after 7 cycles of sequential reactions inaccordance with an embodiment this invention in which a formedpolypeptide is degraded in a controled manner to produce a mixturecontaining a peptide ladder.

[0019]FIG. 8 is the spectrum of the peptide ladder in the region 87-67obtained from the mixture 99-67 in Example 2.

[0020]FIG. 9 is the spectrum of the mixture 66-33 obtained in Example 2.

[0021]FIG. 10 is a spectrum of the low mass region obtained from themixture 66-33 obtained in Example 2 showing the side reaction productsformed during the synthesis of HIV-1 protease.

[0022]FIG. 11 is a spectrum of the reaction mixture obtained in Example3.

[0023]FIGS. 12A and 12B show the reaction support system employed in anembodiment of the inventions which permits multiple simultaneoussequencing of polypeptides.

[0024]FIGS. 13A and 13B are the mass spectra of the peptide laddersformed from both phosphorylated (12A) and unphosphorylated (12B) 16residue peptides containing a serine residue.

[0025]FIG. 14 shows the spectrum of a protein ladder generated byincomplete Edman degradation.

[0026]FIG. 15 shows the spectrum of the mixture obtained in Example 4.

[0027] As will be explained in more detail below, FIGS. 8 through 10 arespectra obtained in the sequencing of a forming polypeptide employingthe process of this invention.

[0028] The invention will be more easily understood if certain of theterms used in this specification and claims are defined.

[0029] The term “polypeptide” is used herein in a generic sense todescribe both high and low molecular weight products comprising linearcovalent polymers of amino acid residues. As the description of thisinvention proceeds, it will be seen that mixtures are produced which maycontain individual components containing 100 or more amino acid residuesor as few as one or two such residues. Conventionally, such lowmolecular weight products would be referred to a amino acids,dipeptides, tripeptides, etc. However, for convenience herein, all suchproducts will be referred to as polypeptides since the mixtures whichare prepared for mass spectrometric analysis contain such componentstogether with products of sufficiently high molecular weight to beconventionally identified as polypeptides.

[0030] The term “formed polypeptide” refers to an existing polypeptidewhich is to be sequenced. It refers, for example to [Glu¹]fibrinopeptideB which is sequenced for purposes of illustration in Example 1. Theprocess of the invention is, of course, most useful for sequencing theprimary structure of unknown polypeptides isolated, for example, byreverse phase HPLC of an enzymatic digest from a protein.

[0031] The term “forming polypeptide” refers to such polypeptides asthey are being formed for example by solid phase synthesis asillustrated in Example 2.

[0032] The term “peptide ladder” refers to a mixture containing a seriesof polypeptides produced by the processes described herein either from aformed or a forming polypeptide. As will be seen from the variousfigures and understood from this description of the invention, a peptideladder comprises a mixture of polypeptides in which the variouscomponents of the mixture differ from the next adjacent member of theseries by the molecular mass of one amino acid residue.

[0033] A “coupling reagents” is a reactant which forms a reactionproduct with a terminal amino acid residue of a polypeptide to besequenced and is subsequently removed together with the residue.

[0034] A “terminating reagent” is a reactant which similarly forms areaction product with a terminal amino acid of polypeptide and is stableto subsequent cycling procedures.

DETAILED DESCRIPTION OF THE INVENTION

[0035] There are several procedures for building peptide ladders, someapplicable to the sequencing of formed polypeptides, others tosequencing of polypeptides as they are being formed.

[0036] One such process will be understood from a study of FIG. 3 whichshows an embodiment of the invention which is applicable to formedpolypeptides. The figure shows the sequencing of an original formedpolypeptide which may contain any number of amino acid residues, even asmany as 50 or more. The polypeptide is shown here by way of illustrationas containing three residues, each residue with a side chain representedby R₁, R₂ or R₃ in accordance with conventional practice.

[0037] The significant feature of this embodiment of the invention, asillustrated in the figure, is that the reaction conditions are cycled toproduce a peptide ladder in the final mixture. The final mixture isanalyzed by mass spectroscopy to determine the exact mass of thecomponents of the ladder, thereby to accumulate the informationnecessary to sequence the original polypeptide.

[0038] The skilled artisan will recognize that this procedure ofsequencing a formed polypeptide makes use of degradation chemistry, butis based on a new principle, i.e. the original polypeptide is employedto generate a family of fragments, each differing by a single amino acidas shown in FIG. 1 wherein X represents a terminating agent. Typically Xwill be a terminating agent that is resistant to all subsequentreactions or manipulations in the cyclic degradation process of thisinvention. As will be described below, in connection with anotherembodiment of this invention, X may also be hydrogen.

[0039] In the process illustrated in FIG. 3, PITC is the couplingreagent and PIC is the terminating reagent. From such a family orpeptide ladder of terminated molecular species prepared as outlined inthe figure, the amino acid sequence can be simply read out in a singlemass spectrometry operation, based on the mass differences between theintact molecular ions. Furthermore, because of the sensitivity of modernmass spectrometers, the accuracy of the amino acid sequence thusdetermined is unaffected, over a wide range (5-fold or more), by theamount of each molecular species present in the mixture.

[0040]FIG. 2 shows an idealized mass spectrum of a peptide ladder inwhich each peak is representative of one member of a series ofterminated polypeptides each member of which differs from the adjacentmember by one amino acid residue.

[0041] Thus, for example, if the peaks of the highest mass in FIG. 2represent a polypeptide, the first five members of which at the aminoterminal end may be:

[0042] Gly¹-Leu-Val-Phe-Ala⁵-,

[0043] the next peak of lower mass would represent

[0044] Leu²-Val-Phe-Ala⁵-

[0045] Subsequent peaks would represent products with one less aminoacid residue. The difference in mass between adjacent members of theseries would be indicative of the amino acid residue removed. Thedifference in molecular mass between the first product on the right andthe adjacent product would correspond to a glycine residue. Subsequentpeaks show the sequential removal of leucine, valine, phenylalanine andalanine residues thus establishing the sequence of these amino acidresidues in the original polypeptide.

[0046]FIG. 3 illustrates a practical sequence of reactions by which theidealized procedure of FIGS. 1 and 2 can be conducted utilizing PITC andPIC as the reagents for sequencing an original formed polypeptide bycycling reaction conditions to produce a peptide ladder forspectrometric analysis.

[0047] In the first step of the sequencing procedure the originalpolypeptide is reacted with a mixture of PITC and PIC under basicconditions. A large molar excess of each reagent is employed. A muchlarger amount of PITC than of PIC is utilized so as to be certain thatat each cycle of the procedure most of the available polypeptide reactswith the coupling agent but that a small measurable fraction of theavailable peptide reacts with the terminating reagent. The fractionreacted with the terminating agent will be determined by the relativeactivities of the coupling agent and the terminating agent, and themolar ratio of the two reagents.

[0048] The first reaction products which form during the basic step ofthe cycle comprise a mixture of original polypeptide terminated with PIC(PC-polypeptide) and an original polypeptide terminated with PITC(PTC-polypeptide). The PIC terminated polypeptide (PC-polypeptide) isstable or essentially stable under all subsequent reaction conditionswith the result that it will be present in a measureable amount in thefinal mixture when that mixture is ready for analysis.

[0049] The next step in the procedure is to subject thePTC-polypeptide/PC-polypeptide mixture to acid conditions whereupon areaction product separates from the PTC-polypeptide. This reactionproduct contains the terminal amino acid residue of the originalpeptide. The separation of this product results in the formation of anew polypeptide which, because the terminal amino acid has been cleavedcontains one less amino acid than the original polypeptide.

[0050] The reaction mixture formed at the end of this cycle contains asthe principal products:

[0051] 1. unreacted coupling and terminating reagents,

[0052] 2. a first reaction product which is the reaction product betweenthe original polypeptide and the terminating reagent. It is a PCterminated polypeptide (PC-polypeptide).

[0053] 3. a new polypeptide from which the amino terminal amino acidresidue has been removed.

[0054] The skilled artisan will readily understand that sequentialrepeats of the cycle just described will result in the formation of amixture which contains as the principal measureable components a seriesof PC-polypeptides each member of which contains one less amino acidresidue than the next higher member of the series. The member of theseries with the highest molecular mass will be the first reactionproduct between the original polypeptide and the terminating reagent.The molecular mass of each subsequent reaction product in the serieswill be the molecular mass of the next higher adjacent member of theseries minus the molecular mass of the terminal amino acid residueremoved by reaction with the PITC. The molecular mass of the PIC,blocking group or any other blocking group selected is irrelevant to thespectrometric analysis since the identity of each amino acid residueremoved from the next adjacent peptide is determined by differences inmolecular mass. These differences identify the amino acid residue, andthe position of that mass difference in the spectrum data set definesthe position of the identified residue in the original polypeptide.

[0055] A constant 5% termination of the available polypeptide at eachcycle for ten cycles of the described chemistry would yield a peptideladder in which the mole fraction of the original polypeptide after eachcycle would be approximately FRACTION MOLE(X)-1-2-3-4-5-6-7-8-9-10-11-12- . . . -n-(OH) .050(X)-2-3-4-5-6-7-8-9-10-11-12- . . . -n-(OH) .048(X)-3-4-5-6-7-8-9-10-11-12- . . . -n-(OH) .045 (X)-4-5-6-7-8-9-10-11-12-. . . -n-(OH) .043 (X)-5-6-7-8-9-10-11-12- . . . -n-(OH) .041(X)-6-7-8-9-10-11-12- . . . -n-(OH) .039 (X)-7-8-9-10-11-12- . . .-n-(OH) .037 (X)-8-9-10-11-12- . . . -n-(OH) .035 (X)-9-10-11-12- . . .-n-(OH) .033 (X)-10-11-12- . . . -n-(OH) .031 (X)-11-12- . . . -n-(OH).60  remains

[0056] The differences in molecular mass between each successive memberof the series in the peptide ladder can be readily determined with highprecision by mass spectroscopy.

[0057] With relatively low molecular weight polypeptides, it is possibleto repeat each cycle without removal of unreacted PITC or PIC. However,as illustrated in Example 1, it is generally preferred to removeunreacted coupling and terminating reagents at the completion of eachcycle. Such removal may also include removal of the cleavage reactionproduct between the coupling reagent and the terminal amino acid.

[0058]FIG. 4 is a more precise summary of the procedure illustrated inFIG. 3 and described in detail above. It specifically illustrates theprocess utilizing a “one pot” technique. In the figure “AA” stands foramino acid and ATZ represents 5-anilinothiazolinone. The other symbolshave the same meaning as above.

[0059] The figure illustrates the preparation of a peptide ladder from aformed polypeptide using controlled ladder-generating chemistry. Thestepwise degradation is conducted with a small amount of PIC and a majorproportion of PITC. Successive cycles of peptide ladder generatingchemistry are performed as described above without intermediateisolation or analysis of released amino acid derivatives. Finally themixture containing the peptide ladder is read out in one step by laserdesorption time-of-flight mass spectrometry (LDMS).

[0060] The coupling and terminating reagents are not limited to the pairdescribed above. Those skilled in the art can readily select otherequivalent reagents. Of course, the procedure can be adapted to eitherthe amino terminal or the carboxy terminal of the polypeptide underanalysis.

[0061] Another procedure for constructing a peptide ladder from a formedpolypeptide is to conduct each cycle in a manner to insure incompletetermination. The process is similar to the above described procedureexcept that only a coupling reagent is employed and the peptide laddercomprises a series of polypeptides none of which is terminated with aterminating reagent but each of which differs from the adjacent memberof the series by one amino acid residue. In this procedure, X of FIG. 1is hydrogen. The principle of this embodiment of the invention is thatonly the coupling reagent is employed in the cycle, and the extent ofreaction is limited for example by limiting reaction times so that allof the original formed polypeptide does not react. As a result, afterthe cycle has been moved to the acid step, the reaction mixture producedwill contain:

[0062] 1. Unreacted PITC,

[0063] 2. The reaction product of PITC and the terminal amino acidresidue with which it has reacted (PTC-polypeptide),

[0064] 3. Unreacted original formed polypeptide,

[0065] 4. A polypeptide with one less amino acid residue than theoriginal polypeptide.

[0066] It will be apparent that by suitable adjustment of reactionconditions, continued repetition of the cycle any selected number oftimes will produce a desired peptide ladder similar to the ladderproduced in the procedure which employs both coupling and terminatingreagents except that the polypeptide members of the ladder are not endblocked with a terminating reagent. This process is similarly applicableto a mixture of polypeptides.

[0067] Another procedure for generating a peptide ladder with only onereagent involves termination by side reaction. In one such process, PITCis employed as a coupling reagent; and, under controlled conditions ofoxidation, a small amount of PITC terminated polypeptide is converted tostable PIC terminated peptide to form a peptide ladder after a selectednumber of cycles. The key to this aspect of the invention is thecontrolled oxidation of a small amount of the PITC terminatedpolypeptide to form PIC terminated polypeptide which is stable, oressentially stable, under subsequent reactions conditions.

[0068] To describe the process with more specificity, the reaction stepsare as follows:

[0069] 1. React the polypeptide to be sequenced under basic conditionswith an excess of PITC to convert substantially all of the polypeptideto PITC terminated polypeptide (PTC-polypeptide).

[0070] 2. React the PTC-polypeptide with a controlled amount of oxygento convert a small portion of the PTC-polypeptide, say 5%, toPC-polypeptide while leaving the balance unchanged.

[0071] 3. Cycle the mixture to the acid step to cleave the PITC boundterminal amino acid from the PTC-polypeptide and leave a polypeptidewith one less amino acid residue than the original polypeptide.

[0072] 4. Repeat the cycle any selected number of times to generate apeptide ladder for mass spectrometric analysis.

[0073] A very significant practical advantage of the process of thisinvention is that it is possible to sequence a plurality of peptides inone reaction system. This advantage arises principally from the highdegree of accuracy that is possible because of the recent advances inmass spectroscopy.

[0074] This aspect of the invention will be understood by reference toFIGS. 12A and 12B which show a suitable device for producing a pluralityof peptide ladders. In the figure, 1 is a reaction support member shownin the form of a cylinder with a holding basin 2 and a through bore 3permitting the passage of chemicals. A series of absorbent members ordiscs 4, for example absorbent membranes are supported by a thin filtermember 5 which may be simply a glass fiber or other suitable filtermaterial.

[0075] In practice, the support member would be in a closed systemadapted to permit the appropriate reactants for the preparation of apeptide ladder on each disc to contact each polypeptide to be sequenced.After each step of the cycle, the reactants exit the support memberthrough the bore 3. The reactants are delivered to the reaction zone byany conventional pumping system of the type employed to collectreactants from a series of reservoirs, mix them and pass the mixturethrough a delivery nozzle.

[0076] Sequencing of formed polypeptides on samples immobilized on asolid support, as in the this embodiment of the invention is especiallyadvantageous because it is applicable to very small amounts of totalsample and because there are reduced handling losses and increasedrecoveries.

[0077] As applied to the system illustrated in the figures, anyconvenient number of polypeptides to be sequenced are separatelyabsorbed on separate discs 4 which may be, for example, an absorbentmembrane such as the cationic, hydrophilic, charge modifiedpolyvinylidene fluoride membrane available from Millipore Corp. asImobilon CD.

[0078] The discs are spaced apart on the filter paper 5 which issupported over the through bore 3 on support member 1 which is thenplaced in a closed system to conduct the controlled cyclic reactionsappropriate to the production of a peptide ladder in accordance withthis invention.

[0079] The amount of polypeptide absorbed on each segment may be assmall as one picomole or even less. Generally, it is from about 1 toabout 10 picomoles.

[0080] In a typical operation, 1 to 10 picomoles of each polypeptide areseparately absorbed on the selected membrane discs and placed separatelyon the filter paper which is then placed on the support member as shown.The peptides are subjected to the PITC/PIC/base/acid cycle describedabove to generate a peptide ladder on each disc. Each separate peptideladder containing mixture to be analyzed may be extracted from eachseparate membrane with an organic solvent containing a small amount ofsurfactant. One useful extraction solvent is 2.5% trifluoroacetic acidin a 1:1 mixture of acetonitrile and 1-o-n-octyl-β-glucopyranoside.

[0081]FIG. 14 shows the spectrum obtained using the absorbent membranetechnology coupled with incomplete termination described above. Togenerate the peptide ladder which was analyzed, 50 picomoles of [Glu-1]fibrinopeptide B on Immobilon-CD membrane was applied to ABI-471Aprotein sequencer (Applied Biosystem). The sequencer was programmedusing 5.5 minute cycle time with a cartridge temperature of 56° C. so asto insure incomplete reaction at each cycle. Six cycles were performed.Under these conditions, a reaction yield of about 56% was estimated. Theresulting peptide ladder is comprised of free N-terminal amines.

[0082] This example illustrates the speed with which the sequencing canbe performed. Similar spectra were obtained with a total loading of only1 picomole of polypeptide on the membrane.

[0083] Although this multiple, simultaneous, sequence analysis ofseparate formed polypeptides utilizing the same chemical reagents forseparate reactions with the said polypeptides has been specificallydescribed by reference to the use of a mixture of specific coupling andterminating reagents in the same reaction zone, it will be apparent thatthe process is equally applicable to the other processes describedabove.

[0084] The system is, of course, applicable to the use of only one discfor the sequencing of a polypeptide or polypeptide mixture.

[0085] Although the discs are shown separately on the support, they mayalso be stacked or replaced with a column of suitably absorbent packingmaterials.

[0086] Further, there may be a number of support members in one deviceand the chemicals fed to the separate support members through a manifoldsystem so that instead of only one reaction zone, there may be aplurality of reaction zones to still further increase the number ofpolypeptides which can be simultaneously sequenced.

[0087] An especially important embodiment of this invention is that itprovides a method of locating covalent modifications on a polypeptidechain particularly post translational modifications of biologicallyimportant products which on chemical or enzymatic hydrolysis producepolypeptides which are phosphorylated, aceylated, glycosylated,cross-linked by disulfide bonds or otherwise modified. Such polypeptidesare referred to in this specification and claims as “modifiedpolypeptides”.

[0088] The inability to directly identify, locate, and quantify modifiedamino acid residues such as phosphorylated residues in a modifiedpolypeptide is a major shortcoming of standard sequencing methods, andhas imposed major limitations on currently important areas of biologicalresearch, such as mechanisms of signal transduction. The process of thisinvention has general application to the direct identification ofpost-translation modifications present in a peptide chain beingsequenced. A modified amino acid residue that is stable to theconditions used in generating the peptide ladder from a formed peptidereveals itself as an additional mass difference at the site of thecovalent modification. As described above, from the mass difference,both the position in the amino acid sequence and the mass of themodified amino acid can be determined. The data generated can provideunambiguous identification of the chemical nature of the posttranslational modification.

[0089] A typical example of this aspect of the invention is the analysisof both phosphorylated and unphosphorylated forms of the 16 residuepeptide LRRASGLIYNNTLMAR amide prepared by the method of Schnolzer et al(9) containing a phosphorylated serine residue prepared by enzymaticreaction using 3′, 5′-cyclic AMP-dependent kinase. After ten cycles ofPITC/PIC chemistry on each form of the peptide using the proceduresdescribed above and illustrated in Example 1, the two separatesequence-defining fragment mixtures (peptide ladders) were each read outby laser desorption mass spectrometry. The resulting protein ladder datasets are shown in FIGS. 13A and 13B. Again, the mass differences definethe identity and order of the amino acids. For the phosphopeptide (FIG.13A), a mass difference of 166.7 daltons was observed for the fifthamino acid from the N-terminal, compared with the mass difference of87.0 for the same residue in the unphosphorylated peptide (FIG. 13B).This measured mass difference corresponds to a phosphyorylated serineresidue, calculated mass 167.1 daltons. Thus, the protein laddersequencing method has directly identified and located a Ser(Pi) atposition five in the peptide. There was no detectable loss of phosphatefrom the phosphoserine residue, which has been regarded in the art asthe most sensitive and unstable of the phosphorylated amino acids.

[0090] Altough only ten cycles of ladder generating chemistry wereperformed, sequence-defining fragments corresponding to eleven residueswere observed, apparently arising from a small amount of prematurecleavage (10). This side reaction which can have serious consequencesfor standard Edman methods, has no effect on the ladder sequencingapproach.

[0091] A specific and very important advantage of this invention is thatit is not limited to analysis of one polypeptide. Mixtures ofpolypeptides can be analyzed simultaneously in one reaction vessel. Eachpolypeptide will give a separate spectrum as shown in idealized form inFIG. 4. In this figure, the molecular masses of the original componentsof the mixture differ by any arbitrary mass difference. Each of theseparate spectra can be analyzed as described above even though theremay be appreciable overlapping in molecular mass among the polypeptidesto be sequenced. This will be clear from the figure. As a result, it ispossible to sequence proteins by analyzing mixtures of polypeptidesobtained by chemical or enzymatic hydrolysis of the protein. The processcan be outlined as follows:

[0092] In most cases, gel electrophoresis will be employed to separateproteins and HPLC to separate polypeptides. Thus, for example, a proteinmixture can be separated into its protein components by electrophoresisand each separate component sequenced by digestion into polypeptides,separation and ladder sequencing in accordance with the process of thisinvention to yield data from which the sequence of the entire proteincan be deduced. The process of the invention may also be employed toobtain extensive data relating to the primary structure of intactproteins at their amino or carboxy terminals.

[0093] There follows a description of the application of this inventionto a forming peptide.

[0094] Stepwise solid phase peptide synthesis involves the assembly of aprotected peptide chain by repetition of a series of chemical steps (the“synthetic cycle”) which results in the addition of one amino acidresidue to an amino acid or peptide chain bound to a support, usually arsin such as methylbenzhydrylamine. The final polypeptide chain is builtup one residue at a time, usually from the C-terminal, by repetition ofthe synthetic cycle. As is well known to peptide chemists, the solidphase synthetic method does not always proceed according to plan. Forany of a number of reasons, some of the polypeptide formed may terminatebefore the final product is produced. For example, a synthesis designedto produce a polypeptide containing twenty amino acid residues mayproduce as side products a variety of polypeptides containing lessernumbers of amino acid residues, e.g. tripeptides, octapeptides anddodecapeptides.

[0095] To utilize the advantages of this invention in solid phasesynthesis, polypeptide-resin samples are collected after each cycle ofamino acid addition. Mixing approximately equal amounts of all samplesobtained in the course of a synthesis yields a peptide ladder containingall possible lengths of resin bound polypeptide. Cleavage of the resinfrom such a mixture produces a mixture of free polypeptide chains of allpossible lengths containing a common carboxy or amino terminal. Usually,stepwise solid phase synthesis proceeds starting from the carboxyterminal. In these cases, the resulting peptide ladder will containpolypeptides all having a common carboxy terminal.

[0096] Consideration of the steps involved in the production of aheptapeptide will explain the procedure. If the heptapeptide to beproduced is of the structure:

[0097] Ala¹-Val-Gly-Leu-Phe-Ala-Gly⁷, the first synthetic step is theattachment of Gly to the resin, usually with a spacer molecule betweenthe resin and the Gly. The next step is the attachment of Nα-blocked Alato the Gly following well known, coupling and deblocking procedures sothat the synthesis is controlled. The cycle is repeated to form theheptapeptide on the resin from which it may be isolated by standardmethods.

[0098] In accordance with the procedure of this invention, a smallsample of polypeptide attached to resin is removed after each cycle.After completion of the synthesis, the seven samples are added togetherto produce a peptide ladder which contains the following components.                        Gly-Resin                     Ala-Gly-Resin                Phe-Ala-Gly-Resin             Leu-Phe-Ala-Gly-Resin        Gly-Leu-Phe-Ala-Gly-Resin     Val-Gly-Leu-Phe-Ala-Gly-ResinAla-Val-Gly-Leu-Phe-Ala-Gly-Resin

[0099] The mixture is then treated, for example with hydrogen fluorideto generate a resin-free peptide ladder which is analyzed massspectrometrically to assure that the final heptapeptide is of thedesired amino acid structure.

[0100] One possible type of side reaction in stepwise solid phasesynthesis is low level blocking at a particular residue (step) in thesynthesis.

[0101] It will be apparent that each has occurred and mixed separatesample collected subsequent to the step at which a side reaction such aslow level blocking has occurred above during the assembly of the finalpolypeptide will contain a portion of such terminated side product withthe result that the amount of such terminated peptide is amplified inthe final mixture as prepared for mass spectrometric analysis. Thus, forexample, if for some reason such as low level blocking there was atermination of some polypeptide at the decapeptide stage in a synthesisdesigned to produce a 20-residue polypeptide, the sample from eachsubsequent synthetic cycle would contain terminated decapeptide and thefinal analytical sample would contain a 10-fold amplification of thisside product. The information obtained by this method of analysis isvery useful in designing optimum procedures for synthesizingpolypeptides, especially those of high molecular weight. One adaptationof this invention to solid phase synthesis is illustrated in Example 2.

[0102] Optionally, the peptide resin samples collected as describedabove may be assayed calorimetrically, for example by a ninhydrinprocedure to determine reaction yields prior to mixing to form a peptideladder. This procedure provides a complimentary method of controllingand assessing the process.

[0103] In the foregoing process, a sample of polypeptide attached to theresin is collected at each step of the synthetic cycle for thepreparation of the final analytical mixture. An alternative procedurefor preparing the final sample is deliberate termination of a smallportion of the forming peptide at each step of the synthetic cyclefollowed by removal of all of the peptides from the resin to form theanalytical mixture directly.

[0104] This can be accomplished by utilizing, instead of one reversiblyblocked amino acid residue at each step in the cycle, a mixture of theselected amino acid residue one portion of which is stable under thereaction conditions, another portion of which is susceptible to removalof the blocking group under controlled conditions.

[0105] If, for example, the amino acid residue to be added to theforming polypeptide is alanine, the peptide bond could be formedutilizing a mixture of Boc-alanine and Fmoc-alanine in which thecarboxyl group is in the appropriate form for reaction, for example inthe form of an hydroxybenzotriazole ester. After the peptide bond hasbeen formed, one of the blocking groups, the removable group, can beremoved under conditions such that the other blocking group remainsintact. Repetition of this cycle will result in the formation of thedesired polypeptide on the resin together with a peptide laddercomprising a series of polypeptides each member of which is joined tothe resin and is terminated by the selected blocking group.

[0106] The procedure will be more readily understood by reference to thepreparation of a specific polypeptide such as:

[0107] Gly¹-Phe-Ala-Leu-Ile⁵.

[0108] The chemistry involved in the preparation of such pentapeptide isstandard solid phase polypeptide synthesis applied in such a manner asto produce a peptide ladder. As applied to this invention, by way ofexample, the C-terminal amino acid residue would be joined to the resin,typically through a linker, as a mixture containing a major proportionof t-Boc-isoleucine and a minor proportion of Fmoc-isoleucine, e.g. in a19:1 ratio.

[0109] The t-Boc blocking group is next removed with an acid such astrifluoroacetic acid. Since the Fmoc group is stable under acidconditions the Fmoc-isoleucine attached to the resin will retain itsblocking group and will be stable to all subsequent reactions.

[0110] In the next step of this synthesis, a 19:1 mixture of Boc-leucineand Fmoc-leucine will be joined to the Ile-Resin, and the Boc blockinggroup selectively removed under acid conditions. As a result of thisstep in the synthetic cycle, the state of the resin may be indicated by:    Fmoc-Ile-Resin Fmoc-Leu-Ile-Resin      Leu-Ile-Resin

[0111] Repetition of these reactions will result in a final resinmixture comprising a peptide ladder which may be represented by:                Fmoc-Ile-Resin             Fmoc-Leu-Ile-Resin        Fmoc-Ala-Leu-Ile-Resin     Fmoc-Phe-Ala-Leu-Ile-ResinFmoc-Gly-Phe-Ala-Leu-Ile-Resin      Gly-Phe-Ala-Leu-Ile-Resin

[0112] This peptide mixture is removed from the resin by standard solidphase procedures which, optionally, will also remove the Fmoc group toproduce an analytical sample ready for analysis by mass spectroscopy asdescribed above.

[0113] The peptide ladder can also be formed by the reverse procedure ofemploying Fmoc as the removable group and t-Boc as the terminatinggroup.

[0114] The adaptation of this invention to solid phase synthesistechniques is illustrated in Example 3 and FIG. 11

[0115] Any blocking group stable to the conditions of chain assemblysynthesis can be used in this application of the invention. For example,acetic acid could be added to each reversibly N-protected amino acid ina stepwise solid phase synthesis in an amount suitable to cause a fewpercent permanent blocking of the growing peptide chain at each step ofthe synthesis. The mass of the blocking group is without effect on theability to read out the sequence of the peptide synthesized since thereadout relies on mass differences between adjacent members of thepolypeptide series as described above.

[0116] Using the procedures described, each individual resin beadcarries the mixture of target full-length peptide and the peptideladder. Typically each bead carries from 1 to 10 or more picomoles ofpolypeptides. Thus, cleavage of the products from a single bead permitsthe direct determination of the sequence of the polypeptide on thatbead.

[0117] It is recognized that the foregoing procedures are described inan idealized form which does not include possible interference by otherfunctional groups such as the hydroxyl group in tyrosine and serine, the“extra” carboxyl groups in dicarboxylic amino acids or the “extra” aminogroups in dibasic amino acids. This method of description has beenadopted to avoid unnecessarily lengthening the specification. Theartisan will recognize the problems which will be introduced by theother functional groups and will know how to deal with them utilizingtechniques well known to peptide chemists.

[0118] It will also be recognized that the procedures described havebeen applied to relatively small polypeptides. They are equallyapplicable to large polypeptides. For example, if the formingpolypeptide is one which contains twenty or more amino acid residues, itmay be expedient to sequence the pentapeptide, the decapeptide and thepentadecapeptide to be certain that the synthesis is going according toplan.

[0119] A variety of other chemical reaction systems can be employed togenerate peptide ladders for analysis in accordance with this invention.

[0120] It will be recognized that there are a number of significantadvantages to the processes of this invention. For example, the demandson yield of the chemical degradation reactions are much less stringentand more readily achieved than by wet chemical stepwise degradationtechniques such as the Edman degradation in which low molecular weightderivatives are recovered and analyzed at each chemical step. Otheradvantages include accuracy, speed, convenience, sample recovery, andthe ability to recognize modifications in the peptide such asphosphorylation. Relatively unsophisticated and inexpensive massspectrometric equipment, e.g. time of flight; single quadrupole; etc.can be used.

[0121] By employing the process of this invention, it is routinelypossible to sequence polypeptides containing 10 or more amino acidresidues from one picomole, or even a smaller amount of a polypeptide inone hour or less including cyclic degradation, mass spectrometry, andinterpretation.

[0122] The processes described may be readily automated i.e., carriedout for example in microtiter plates, using an x, y, z chemical robot.Furthermore, the determination of amino acid sequence from massspectrometric data obtained from the protein sequencing ladders isreadily carried out by simple computer algorithms. The process of theinvention therefore includes computer read-out of the spectra of thepeptide ladders produced.

[0123] The skilled artisan will recognize that there are somelimitations to the process of the invention as described above.

[0124] For example, some pairs of amino acids such as leucine andisoleucine have the same molecular weights. Therefore, they can not bedistinguished by mass differences of terminated polypeptides in aseries. There are several procedures for avoiding this difficulty. Oneis to differentiate them by cDNA sequencing. They are highly degeneratecodons, so they can be accommodated by inosine substitution in DNAprobes/primers for isolation/identification of the corresponding gene.This limitation will have little impact on practical application of theinvention.

[0125] Further, several amino acids differ by only 1 amu. This placesstringent requirements on accuracy of mass determination. However, thisinvention utilizes a determination of mass differences between adjacentpeaks, not a determination of absolute masses. Since mass differencescan be determined with great accuracy by mass spectroscopy, thelimitation will also be of little practical significance.

[0126] Finally, samples which are blocked at the amino or carboxyterminal may not be susceptible to the generation of peptide ladders.This problem can be circumvented by chemical or enzymatic fragmentationof the blocked polypeptide chain to yield unblocked segments which canbe separately analyzed.

[0127] The following non-limiting examples are given by way ofillustration only and are not to be considered as limitations of theinvention many apparent variations of which may be made withoutdeparting from the spirit or scope thereof.

EXAMPLE 1 Sequencing of [Glu¹]Fibrinopeptide B

[0128] [Glu¹]Fibrinopeptide B was purchased from Sigma Chemical Co. (St.Louis, Mo.). The reported sequence was:Glu¹-Gly-Val-Asn-Asp⁵-Asn-Glu-Glu-Gly-Phe¹⁰-Phe-Ser-Ala-Arg¹⁴. Matrixassisted laser desorption mass spectrometry gave MW 1570.6 dalton(Calculated: 1570.8 dalton) and showed high purity of the startingpeptide. A mixture of PITC plus 5% v/v phenylisocyanate PIC was used inthe coupling step. PIC reacts with the NH₂-of a polypeptide chain toyield an Nα-phenylcarbamyl-peptide which is stable to the conditions ofthe Edman degradation. A modification of a standard manual Edmandegradation procedure (6) was used. All reactions were carried out inthe same 0.5 mL polypropylene microfuge tube under a blanket of drynitrogen. Peptide (200 pmoles to 10 nmole) was dissolved in 20 ul ofpyridine/water (1:1 v/v; pH10.1); 20 uL of coupling reagent containingPITC:PIC:pyridine:hexafluoroisopropanol (20:1:76:4 v/v) was added to thereaction vial. The coupling reaction was allowed to proceed at 50° C.for 3 minutes. The coupling reagents and non-peptide coproducts wereextracted by addition of 300 uL of heptane:ethyl acetate (10:1 v/v),gentle vortexing, followed by centrifugation to separate the phases. Theupper phase was aspirated and discarded. This washing procedure wasrepeated once, followed by washing twice with heptane:ethyl acetate (2:1v/v). The remaining solution containing the peptide products was driedon a vacuum centrifuge. The cleavage step was carried out by addition of20 uL of anhydrous trifluoroacetic acid to the dry residue in thereaction vial and reaction at 50° C. for 2 minutes, followed by dryingon a vacuum centrifuge. Coupling-wash-cleavage steps were repeated for apredetermined number of cycles. The low MW ATZ/PTH derivatives releasedat each cycle were not separated/analyzed. Finally, the total productmixture was subjected to an additional treatment with PIC to convert anyremaining unblocked peptides to their phenylcarbamyl derivatives. Inthis final step, the sample was dissolved in 20 uL oftrimethylamine/water (25% wt/wt) in pyridine (1:1 v/v); 20 uL ofPIC/pyridine/HFIP (1:76:4 v/v) was added to the reaction vial. Thecoupling reaction was carried out at 50° C. for 5 min. The reagents wereextracted as described above. After the last cycle of ladder generatingchemistry, the product mixture was dissolved in 0.1% aqueoustrifluoroacetic acid: acetonitrile (2:1, v/v). A 1 uL aliquot (250 pmoltotal peptide, assuming no losses) was mixed with 9 uL ofα-cyano-4-hydroxy-cinnammic acid (5 g/L in 0.1% trifluoroacetic acid:acetonitrile, 2:1 v/v), and 1.0 uL of this mixture of total peptideproducts (25 pmol) and matrix was applied to the probe tip and dried ina stream of air at room temperature. Mass spectra were acquired inpositive ion mode using a laser desorption time-of-flight instrumentconstructed at The Rockefeller University (7). The spectra resultingfrom 200 pulses at a wavelength of 355 nm, 15 mJ per pulse, wereacquired over 80 seconds and added to give a mass spectrum of theprotein sequencing ladder shown in FIG. 7. Masses were calculated usingmatrix peaks of known mass as calibrants.

[0129] Peptide sequence read-out. Positive ion (MALDMS) spectra of[Glu¹]Fibrinopeptide B is shown in FIG. 6. A protonated molecular ion[M+H] was observed at m/z 1572.5 (calculated value is 1571.8).

[0130] Its positive ion MALDMS spectrum of the reaction mixture obtainedafter seven cycles is shown in FIG. 6. Each of the peaks in the spectrumrepresents a related phenylcarbamoylpeptide derivative in the peptideladder (except a few peaks which will discussed later). The amino acidsequence can be easily read-out from the mass difference of adjacent twopeaks for instance, the mass difference are 129.1, 56.9, and 99.2between peaks at m/z 1690.9 and 1561.8, peaks at m/z 1561.8 and 1504.9and peaks at m/z 1504.9 and 1405.7. Which correspond to glutamic acid(ca. 129.12), glycine (ca. 57.05) and valine (ca. 99.13) residues,respectively. One set of paired peaks gives mass difference 119.0(1062.1-943.1) which corresponds to the phenylcarbamoyl group. In otherwords, these two peaks represent one piece of peptide with or withoutphenylcarbamoyl group. Peak at m/z 1553.8 corresponds partially blockedpeptide with pyroglutamic acid at the N-terminus. This results fromcyclization of the N-terminal Glu under the reaction conditions used.Such products are readily identified from the accurately measured massand know chemical reaction tendencies.

EXAMPLE 2

[0131] Stepwise solid phase synthesis of the 99 amino acid residuepolypeptide chain corresponding to the monomer of the HIV-1 protease(SF2 isolate): PQITLWQRPLVTIRIGGQLKEALLDTGADDTVLEEMNLPGKWKPKMIGGIGGFIKVRQYDQIPVEI(Aba)GHKAIGTVLVGPTPVNIIGRNLLTQIG (Aba)TLNF⁹⁹

[0132] [where Aba=α-amino-n-butyric acid] was undertaken. Highlyoptimized Boc-chemistry instrument-assisted stepwise assembly of theprotected peptide chain was carried out on a resin support, according tothe method described by S. B. H. Kent (8). Samples (3-8 mg, about 1umole each) were taken after each cycle of amino acid addition. Theprotected peptide-resin samples were mixed in three batches ofconsecutive samples: (number corresponds to the amino acid after whichsample was taken, i.e. residue number in the target sequence.) 99-67;66-33; 32-1. The first such mixture contained the peptides:                           99-Resin                         98-99-Resin                     97-98-99-Resin                   96-97-98-99-Resin                 . . . (etc.) . . .          70 . . . 96-97-98-99-Resin      69-70 . . . 96-97-98-99-Resin    68-69-70 . . . 96-97-98-99-Resin67-68-69-70 . . . 96-97-98-99-Resin

[0133] Similarly for the other two mixtures. The mixed batches ofpeptide-resin were deprotected and cleaved with HF (1 hours, at 0° C.,plus 5% cresol/5%/thiocresol). The products were precipitated withdiethyl ether, dissolved in acetic acid-water 950/50%, v/v) and thenlyophilized.

[0134] Each peptide mixture was dissolved in 0.1% TFA, 1 uL of thepeptide mixture (10 uM per peptdie component) was added to 9 uL of4-hydroxy- -cyanocinnamic acid in a 1:2 (v/v) ratio of 30%acetonitrile/0.1% aqueous trifluoroacetic acid. 0.5 uL of the resultingmixture was applied to the mass spectrometer probe and inserted into theinstrument (7). The spectra shown in FIGS. 8 and 9 are the result ofadding the data of each of 100 laser shots performed at a rate of 2.5laser shots/second. FIG. 8 shows the mass spectrum obtained from themixture resulting from cleaving mixed samples from residues 99-67 of thesynthesis. FIG. 9 shows the mass spectrum obtained from the mixtureresulting from cleaving mixed samples from residues 66-33 of thesynthesis. Table 1 shows the measured mass differences betweenconsecutive peaks of a selection of these peaks and compares them withthe mass differences calculated from known sequences of the targetpeptides. The agreements are sufficiently close to allow confirmation ofthe correctness of the synthesis.

[0135]FIG. 11 shows mass spectra of the mixture obtained from mixedsamples from residues (66-33) of the synthesis.

[0136] The sequence of the assembled polypeptide chain can be read outin a straightforward fashion from the mass differences betweenconsecutive peaks in the mass spectra of the peptide mixture. Thisconfirmed the sequence of amino acids in the peptide chain actuallysynthesized. The identity of the amino acids as determined by such massdifferences is shown in Table 1 TABLE 1 The identify of amino acid bythe mass differences in protein ladder sequencing using matrix-assistedlaser desorption mass spectrometry. Mass Mass Difference DifferenceAmino (Measured, Amino (Measured, Acid Da) Deviation Acid Da) DeviationLeu³³ 113.3 0.1 Asp⁶⁰ 114.8 −0.3 Glu³⁴ 129.7 0.6 Gln⁶¹ 128.7 0.6 Glu³⁵129.5 0.4 Ile⁶² 113.2 0.0 Met³⁶ 130.8 −0.4 Pro⁶³ 97.0 −0.1 Asn³⁷ 115.00.9 Val⁶⁴ 99.4 0.3 Leu³⁸ 112.4 −0.8 Glu⁶⁵ 128.6 −0.5 Pro³⁹ 97.9 0.8Ile⁶⁶ 113.3 0.1 Gly⁴⁰ 56.1 −0.9 Aba⁶⁷ 84.9 −0.2 Lys⁴¹ 128.1 0.0 Gly⁶⁸57.0 0.0 Trp⁴² 186.4 0.2 His⁶⁹ 137.3 0.2 Lys⁴³ 128.2 0.0 Lys⁷⁰ 127.8−0.4 Pro⁴⁴ 97.1 0.0 Ala⁷¹ 71.4 0.3 Lys⁴⁵ 128.0 −0.2 Ile⁷² 113.4 0.2Met⁴⁶ 131.9 0.7 Gly⁷³ 56.8 −0.2 Ile⁴⁷ 112.6 −0.6 Thr⁷⁴ 101.1 0.0 Gly⁴⁸57.9 0.9 Val⁷⁵ 99.2 0.1 Gly⁴⁹ 56.3 −0.7 Leu⁷⁶ 113.1 −0.1 Ile⁵⁰ 112.4−0.8 Val⁷⁷ 99.1 0.0 Gly⁵¹ 57.6 0.6 Gly⁷⁸ 57.1 0.1 Gly⁵² 57.5 0.5 Pro⁷⁹97.2 0.1 Phe⁵³ 147.3 0.1 Thr⁸⁰ 101.1 0.0 Ile⁵⁴ 112.5 −0.7 Pro⁸¹ 97.1 0.0Lys⁵⁵ 128.9 0.8 Val⁸² 99.2 0.1 Val⁵⁶ 99.0 −0.1 Asn⁸³ 113.8 −0.3 Arg⁵⁷156.2 0.0 Ile⁸⁴ 113.4 0.2 Gln⁵⁸ 128.4 0.3 Ile⁸⁵ 113.1 0.0 Tyr⁵⁹ 162.6−0.6 Gly⁸⁶ 57.1 0.0

[0137] In addition, terminated by-products (where the peptide chain hasbecome blocked and does not grow anymore) are present in everypeptide-resin sample taken after the step in which the block occurred.Thus, there is an amplification factor equal to the number of resinsamples in the batch after the point of termination. This can be seen inFIG. 10 (samples #66-33) which contains a peak at 3339.0. Thiscorresponds to the peptide 71-99, 3242.9 (N-terminal His71) plus 96.1dalton. The characteristics mass, together with knowledge of thechemistry used in the synthesis identifies the blocking group as CF3CO-(97.1-H=96.1 dalton). The observed by product is thetrifluoroacetyl-peptide, Nα-Tfa-(71-99). The ratio of the amount of thiscomponent to the average amount of the other components is about 2:1.There were 34 samples combined in this sample. Thus, the terminatedbyproduct Nα-Tfa-(71-99) had occurred at a level of about 5 mol %. Thisside reaction, specific to the N-terminal His-peptide chain, has notpreviously been reported. This illustrates the important sensitivityadvantage provided by this amplification effect in detecting terminatedpeptides. Such byproducts are not readily detected by any other means.

EXAMPLE 3

[0138] Boc/Fmoc Terminations Synthesis of the peptideLRRAFGLIGNNPLMAR-amide was performed manually on a 0.2 mmol scale usingp-methylbenzhydrylamine resin and 0.8 mmoles amino acid (95 mol %N-α-Boc, 5 molt N-α-Fmoc) according to the in situ neutralizationmethods of Schnolzer et al (9). The following side chain protectinggroups were used: Boc-Arg, tosyl; Fmoc-Arg,2,3,6-trimethyl-4-methoxybenzenesulfonyl (Mtr). Fmoc-Arg(Mtr) was usedfor its greater stability in trifluoroacetic acid (TFA). Aftercompletion of the chain assembly, Fmoc groups were removed using 50%piperidine/DMF, followed by Boc group removal in TFA. The peptidefragments were then cleaved from the resin by treatment with HF-10%p-cresol (0° C., 1 hour). The resulting crude peptide products wereprecipitated and washed with ether, dissolved in 50% acetic acid,diluted with water and lyophilized. The mass spectra of the reactionmixture thus produced is shown in FIG. 11.

EXAMPLE 4

[0139] Post-ninhydrin Experiment The machine-assisted assembly of thepeptide LRRASGLIYNNPLMAR-amide was performed according to the in situneutralization methods of Schnolzer and Kent (9) on a 0.25 mmol scaleusing MBHA resin and 2.2 mmol N—-Boc amino acids. The following sidechain protecting groups were used: Arg, tosyl; Asn, xanthyl; Ser,benzyl(Bzl); Tyr, bromobenzyloxycarbonyl(BrZ). Resin samples werecollected at each step in the synthesis and each sample was individuallysubjected to the quantitative ninhydrin reaction. These samples werethen pooled and the Boc groups removed in neat TFA. Cleavage of thepeptide fragments from the resin was performed by treatment with HF-10%p-cresol (OC, 1 hour). The resulting crude peptide products wereprecipitated and washed with ether, dissolved in 50% acetic acid,diluted with water and lyophillized. The mass spectrum of the mixture isshown in FIG. 15.

Citations

[0140] The following publications are referred to in thisspecifications. The complete disclosure of each of them is herebyincorporated by references.

[0141]1. Aebersold et al, Protein Science 1, 494 (1992)

[0142]2. R. Self, A. Parente, Biomed. Mass Spectrom. 10, 78 (1983)

[0143]3. L. A. Smith, R. M. Caprioli, Biomed. Mass Spectrom. 10, 98(1983)

[0144]4. B. T. Chait, T. Chaudhary, F. H. Field, “Methods in Proteinsequence Analysis 1986”, K. A. Walsh, ed., Humana Press 1987, pp.483-493, and uncontrolled chemical degradation

[0145]5. A. Tsugita, K. Takamoto, M. Kamo, H. Iwadate, Eur. J. Biochem.206, 691 (1992)

[0146]6. G. E. Tarr (1977), in Methods Enzymology 47, 355.

[0147]7. R. C. Beavis and B. T. Chait (1989), Rapid Commun. MassSpectrom. 3, 233.

[0148]8. S. B. H. Kent, Annual Rev. Biochem. 57, 957-984 (1988)

[0149]9. Schnolzer et al, Int. J. Peptide Protein Res. 40, 1992, 180-193

[0150]10. W. A. Schroeder, Meth. Enzymol. 25, 298 (1972)

1 24 20 amino acids amino acid <Unknown> linear peptide Modified-site 1/product= “OTHER” /note= “Xaa = alpha-amino-n-butyric acid” 1 Xaa GlyHis Lys Ala Ile Gly Thr Val Leu Val Gly Pro Thr Pro Val 1 5 10 15 AsnIle Ile Gly 20 18 amino acids amino acid <Unknown> linear peptide 2 GlyIle Gly Gly Phe Ile Lys Val Arg Gln Tyr Asp Gln Ile Pro Val 1 5 10 15Glu Ile 16 amino acids amino acid <Unknown> linear peptide 3 Leu Glu GluMet Asn Leu Pro Gly Lys Trp Lys Pro Lys Met Ile Gly 1 5 10 15 16 aminoacids amino acid <Unknown> linear peptide 4 Leu Arg Arg Ala Phe Gly LeuIle Gly Asn Asn Pro Leu Met Ala Arg 1 5 10 15 11 amino acids amino acid<Unknown> linear peptide Modified-site 5 /product= “OTHER” /note= “Xaa =phosphorylated serine” 5 Leu Arg Arg Ala Xaa Gly Leu Ile Tyr Asn Asn 1 510 11 amino acids amino acid <Unknown> linear peptide 6 Leu Arg Arg AlaSer Gly Leu Ile Tyr Asn Asn 1 5 10 16 amino acids amino acid <Unknown>linear peptide 7 Leu Arg Arg Ala Ser Gly Leu Ile Tyr Asn Asn Pro Leu MetAla Arg 1 5 10 15 5 amino acids amino acid <Unknown> linear peptide 8Gly Leu Val Phe Ala 1 5 4 amino acids amino acid <Unknown> linearpeptide 9 Leu Val Phe Ala 1 16 amino acids amino acid <Unknown> linearpeptide Modified-site 16 /product= “OTHER” /note= “Xaa = argininamide”10 Leu Arg Arg Ala Ser Gly Leu Ile Tyr Asn Asn Thr Leu Met Ala Xaa 1 510 15 7 amino acids amino acid <Unknown> linear peptide 11 Ala Val GlyLeu Phe Ala Gly 1 5 4 amino acids amino acid <Unknown> linear peptideModified-site 4 /product= “OTHER” /note= “Xaa = glycine bound to a resinsuch as methylbenzhydrylamine” 12 Leu Phe Ala Xaa 1 5 amino acids aminoacid <Unknown> linear peptide Modified-site 5 /product= “OTHER” /note=“Xaa = glycine bound to a resin such as methylbenzhydrylamine” 13 GlyLeu Phe Ala Xaa 1 5 6 amino acids amino acid <Unknown> linear peptideModified-site 6 /product= “OTHER” /note= “Xaa = glycine bound to a resinsuch as methylbenzhydrylamine” 14 Val Gly Leu Phe Ala Xaa 1 5 7 aminoacids amino acid <Unknown> linear peptide Modified-site 7 /product=“OTHER” /note= “Xaa = glycine bound to a resin such asmethylbenzhydrylamine” 15 Ala Val Gly Leu Phe Ala Xaa 1 5 5 amino acidsamino acid <Unknown> linear peptide 16 Gly Phe Ala Leu Ile 1 5 4 aminoacids amino acid <Unknown> linear peptide Modified-site 1 /product=“OTHER” /note= “Xaa = 9-fluoromethoxycarbonyl (Fmoc) phenylalanine”Modified-site 4 /product= “OTHER” /note= “Xaa = isoleucine bound to aresin such as methylbenzhydrylamine” 17 Xaa Ala Leu Xaa 1 5 amino acidsamino acid <Unknown> linear peptide Modified-site 1 /product= “OTHER”/note= “Xaa = 9-fluoromethoxycarbonyl (Fmoc) glycine” Modified-site 5/product= “OTHER” /note= “Xaa = isoleucine bound to a resin such asmethylbenzhydrylamine” 18 Xaa Phe Ala Leu Xaa 1 5 5 amino acids aminoacid <Unknown> linear peptide Modified-site 5 /product= “OTHER” /note=“Xaa = isoleucine bound to a resin such as methylbenzhydrylamine” 19 GlyPhe Ala Leu Xaa 1 5 14 amino acids amino acid <Unknown> linear peptide20 Glu Gly Val Asn Asp Asn Glu Glu Gly Phe Phe Ser Ala Arg 1 5 10 99amino acids amino acid <Unknown> linear peptide Modified-site 67/product= “OTHER” /note= “Xaa = alpha-amino-n-butyric acid”Modified-site 95 /product= “OTHER” /note= “Xaa = alpha-amino-n-butyricacid” 21 Pro Gln Ile Thr Leu Trp Gln Arg Pro Leu Val Thr Ile Arg Ile Gly1 5 10 15 Gly Gln Leu Lys Glu Ala Leu Leu Asp Thr Gly Ala Asp Asp ThrVal 20 25 30 Leu Glu Glu Met Asn Leu Pro Gly Lys Trp Lys Pro Lys Met IleGly 35 40 45 Gly Ile Gly Gly Phe Ile Lys Val Arg Gln Tyr Asp Gln Ile ProVal 50 55 60 Glu Ile Xaa Gly His Lys Ala Ile Gly Thr Val Leu Val Gly ProThr 65 70 75 80 Pro Val Asn Ile Ile Gly Arg Asn Leu Leu Thr Gln Ile GlyXaa Thr 85 90 95 Leu Asn Phe 54 amino acids amino acid <Unknown> linearpeptide 22 Leu Glu Glu Met Asn Leu Pro Gly Lys Trp Lys Pro Lys Met IleGly 1 5 10 15 Gly Ile Gly Gly Phe Ile Lys Val Arg Gln Tyr Asp Gln IlePro Val 20 25 30 Glu Ile Xaa Gly His Lys Ala Ile Gly Thr Val Leu Val GlyPro Thr 35 40 45 Pro Val Asn Ile Ile Gly 50 16 amino acids amino acid<Unknown> linear peptide Modified-site 16 /product= “OTHER” /note= “Xaa= argininamide” 23 Leu Arg Arg Ala Phe Gly Leu Ile Gly Asn Asn Pro LeuMet Ala Xaa 1 5 10 15 16 amino acids amino acid <Unknown> linear peptideModified-site 16 /product= “OTHER” /note= “Xaa = argininamide” 24 LeuArg Arg Ala Ser Gly Leu Ile Tyr Asn Asn Pro Leu Met Ala Xaa 1 5 10 15

1.-49. (Canceled) 50.-63. (Canceled)
 64. A method for identifying acovalent modification of an amino acid residue in a polypeptide chaincomprising: detecting a mass difference between a formed polypeptide anda modified polypeptide by mass spectrometry, wherein the modifiedpolypeptide comprises a covalent modification of an amino acid residuein the formed polypeptide, whereby the mass difference identifies thecovalent modification.
 65. The method of claim 64 wherein the covalentmodification is phosphorylation.
 66. The method of claim 64 wherein thecovalent modification is acetylation.
 67. The method of claim 64 whereinthe covalent modification is glycosylation.
 68. The method of claim 64wherein the covalent modification is a disulfide bond.
 69. The method ofclaim 64, wherein said mass spectrometry is ion trap mass spectrometry.70. The method of claim 64, wherein said mass spectrometry is quadripolemass spectrometry.
 71. The method of claim 64 further comprising: (i)producing reaction mixtures from the formed and the modifiedpolypeptide, each reaction mixture containing a peptide laddercomprising a series of adjacent polypeptides in which each member of theseries differs from the next adjacent member by one amino acid residue;(ii) determining the differences in molecular mass between adjacentmembers of each series by mass spectroscopy, said differences coupledwith the positions of said adjacent members in the respective seriesbeing indicative of the identity and position of the amino acid residuein the formed or modified polypeptide; (iii) identifying an amino acidresidue in the modified polypeptide that comprises a covalentmodification wherein the amino acid residue is identified by having anadditional mass difference compared with the corresponding amino acidresidue in the formed polypeptide.
 72. The method of claim 71 whereinproducing the peptide ladders comprises: (1) reacting the formed andmodified polypeptide with a molar excess of a pair of reagentscomprising a coupling reagent and a terminating reagent each of whichforms a reaction product with a terminal amino acid residue of theformed or modified polypeptide to be analyzed under a first reactioncondition; the reaction product generated between the terminatingreagent and the terminal amino acid residue of the formed or modifiedpolypeptide being stable under all subsequent reaction conditions; thereaction product generated between the coupling reagent and terminalamino acid residue of the polypeptide to be analyzed being removable asa cleavage product from the original formed or modified polypeptideunder a second reaction condition; (2) changing the reaction conditionsso that the cleavage product separates, thereby to form a reactionmixture comprising: i. unreacted coupling and terminating reagents, ii.a first reaction product which is the reaction product between theoriginal formed or modified polypeptide and the terminating reagent,iii. a newly formed polypeptide from which the terminal amino acidresidue has been removed; (3) repeating steps (1) and (2) any selectednumber of cycles thereby to form a final mixture which comprises: i.reaction product between the original formed or modified polypeptide andthe terminating reagent, ii. a peptide ladder which is series ofadjacent reaction products which is formed by reaction between theterminating reagent and the terminal amino acid residue of a fraction ofthe newly generated polypeptide of each cycle, and (4) determining thedifferences in molecular mass between adjacent members of the series ofreaction products by mass spectroscopy, said differences being equal tothe molecular mass of the amino acid residue cleaved from the originalformed or modified polypeptide and from each subsequent formed ormodified polypeptide of the series, said differences coupled with thepositions of said adjacent members in the mass spectrum being indicativeof the identity and position of that amino acid residue in the originalformed or modified polypeptide.
 73. The method of claim 72 wherein thecoupling agent is PITC, the terminating agent is PIC, the first set ofreaction conditions comprises basic conditions and the second set ofconditions comprises acidic conditions.
 74. The method of any of claims64, 65, 66, 67, 68, 69, 70, 71, 72, or 73 wherein the formed polypeptideand the modified polypeptide are analyzed simultaneously in a mixture.